Characterization of hnRNP A3 Isoforms in The A2RE-Proteome Ying Wei MSc, MInfTech A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 School of Chemistry and Molecular Biosciences 1 Abstract Polarity is an important prerequisite for cell differentiation. The development of cellular asymmetry and cell polarity can be achieved by either selectively transporting key intracellular proteins or vectorial trafficking mRNAs to a subcellular location for local translation. The “A2RE-mRNA trafficking pathway” fits in the latter category, renowned for moving the myelin basic protein (MBP) RNA molecules from the nucleus to cytoplasmic myelin compartment of oligodendrocytes. This prominent pathway is named after a 11-nucleotide cis-acting sequence of “A2 response element (A2RE)”, which is necessary and sufficient for mRNA localization. The universally expressed protein hnRNP A2 was the first identified trans-acting factor bound to the A2RE cis- element directing the A2RE-containing transcripts to their subcellular locations along cytoskeletal components. In addition to hnRNP A2, hnRNP A3 is the second abundant proteins pulled down by the A2RE- sequence. At the beginning of this project, hnRNP A3 had just been identified as a novel key component along with other A2RE-binding proteins. A lot of structural and functional questions arising from the discovery of hnRNP A3 in the A2RE-context await answers. The detailed structural knowledge of hnRNP A3 among the A2RE-binding proteins was essential prior to the investigation of its biological involvement in the A2RE-trafficking pathway. Therefore, the overall aim of this project was to characterize the structure of hnRNP A3 isoforms which were purified by binding to the A2RE-sequence. At the same time, the structural comparison with other A2RE-binding proteins was also carried out, aiming to find out any similar or distinct features between these proteins when they were associated together with the same A2RE-sequence. The investigations in this project were established from aspects of genomics, proteomics and cellular location to characterize the hnRNP A3 isoforms. The experimental data, presented in this thesis, were collected reproducibly and comprise all above aspects. From the genomic aspect, the two splicing forms of hnRNP A3 in rat brain were exclusively identified through the extensive PCR screening. At the protein level, the masses of the intact A2RE- binding proteins were measured by liquid chromatography mass spectrometry (LC/MS) and analyzed. Next, the rat brain A2RE-binding proteins were successfully separated by two- dimensional gel electrophoresis (2-DE), showing a more complicated profile of hnRNP A/B isoforms in the A2RE-context. Each binding components were identified by mass spectrometry (MS) based peptide mass fingerprinting. 2 Targeting the complexity of hnRNP A3 inside the A2RE-proteome, the post-translational modifications (PTMs) of hnRNP A3 were investigated. Phospho-proteins of the A2RE proteome were detected by blotting and staining. Moreover, the pattern of protein phosphorylation was observed to be isoform-specific between the alternatively spliced isoform pairs. In terms of Arg modifications, the methylation of A3 was confirmed, and six asymmetric dimethylated residues of A3 were successfully identified in the protein Gly-rich region by the bottom-up tandem MS proteomics. The same MS/MS proteomic method also invalidated the occurrence of citrullinated Arg residues in A3. In the developmental study, the uneven levels between two alternatively spliced HNRPA3 transcripts were discovered. From the observation, the truncated A3b transcripts always maintain a stable higher level than the full-length A3a transcripts. The subcellular distribution patterns of the A3 isoforms were also explored by transfecting GFP-tagged A3 vectors into neuronal cells, in comparison with its endogenous cellular pattern detected by the specific immunostaining method. In summary, the investigations of hnRNP A3 in this project expanded from its encoding gene to its final cellular location. With more results acquired, the hnRNP A3 isoforms became more significant along with the other key A2RE-binding components A1 and A2/B1. The clarified 2-D profile of the A2RE proteome and unambiguously identified methylated residues of A3 provide insight into the functionally interweaved hnRNP A/B paralogs. Although the conclusions deduced from this project only represent the hnRNP A3 isoforms inside the A2RE-context, the characterization of A3 isoforms under such specific circumstance still provide valuable information for studies of hnRNP A/B paralogs in other fields, opening up a window for studying downstream mechanisms of mRNA biogenesis and metabolism. 3 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. 4 Publications during candidature Journal Publication Friend, L. R., Landsberg, M. J., Nouwens, A. S., Wei, Y., Rothnagel, J. A., & Smith, R. (2013). Arginine methylation of hnRNP A2 does not directly govern its subcellular localization. PLoS One, 8(9), e75669. doi: 10.1371/journal.pone.0075669 Conference Publication Ying Wei, Alice Ma and Ross Smith. The role of hnRNP A3 isoforms in mRNA trafficking. ComBio 2005, Adelaide, 25 - 29 September, 2005 (Abstract and poster). Ying Wei and Ross Smith. Structural characterization of hnRNP A3 isoforms. ComBio 2006, Brisbane, 24 - 28 September, 2006 (Abstract and poster). Ying Wei and Ross Smith. Subcellular localization of hnRNP A3 isoforms in neural cells. ComBio 2007, Sydney, 22-26 September, 2007 (Abstract and poster). Publications included in this thesis No publications included 5 Contributions by others to the thesis Dr. Amanda Nouwens, contribution in operating and calibrating the MALDI-TOF mass spectrometry system. Statement of parts of the thesis submitted to qualify for the award of another degree None 6 Acknowledgements The project presented in this thesis was carried out in the School of Chemistry and Molecular Biosciences, the University of Queensland, St. Lucia campus. Writing up a 200-page-thesis is a huge work for me, however, the efforts and supports I have received from people around me during my thesis writing will be ten times longer if I write the details down. Coming to this far, I have run out of my words in writing, but full of gratitude and appreciation to all these great people: my supervisors Prof. Ross Smith, Assoc. Prof. Joseph A. Rothnagel and Dr. Lexie Friend; my peer members Dr. Jodie Hatfield, Dr. Yaowu He, Dr. Sergio Sara, Dr. Wolfgan Hofmeister, Yuan Li; and all my family members. I would address my special appreciation to Ross for his mentoring and continuous supports all these years; to Lexie for her caring in both professional and personal areas, her persistence and strong encouragements leading me through all the difficulties. You both let me know this journey to the Ph.D. degree is much more than a scientific research—it is a philosophy and attitude of life. Great thanks for igniting me after years of pending in writing, refreshing me with many innovative brainstorms, believing me without any doubt to complete this IMPOSSIBLE job… Behind my thesis, there are countless conversations, emails and visits: caring about every progress I have made, commenting on every page I have written, passing me every piece of information that might be helpful. Although this thesis could not correctly reflect the efforts you have invested in me, I have tried my best. Sincerely thank you both! To my family, I would like thank my nephews Wesley, baby Neo and niece Minnie. Wesley, your gift in Math and Music sometimes sidetracked me from my research, but your peerless talents were my true pride when I was down. Your academic excellency always urges me to stand higher so as to provide more advanced guidance for you. I am proud of you! Last but not least thanks go to my mum. Your company these days is the best support for me. I love you from the bottom of my heart! 7 Keywords hnRNP, A2RE, isoform, methylation, phosphorylation, proteomics, two-dimensional electrophoresis, mass spectrometry,